Project Summary Neutrophils are primary cells of the innate immune system that are necessary for host defense, however, persistent neutrophil inflammation contributes to tissue damage and chronic inflammation in broad diseases including cardiovascular disease, autoimmune disease and cancer. It was previously thought that resolution of neutrophil inflammation occurred through neutrophil death and macrophage phagocytosis; however, we recently discovered that neutrophils also leave sites of tissue inflammation through a process referred to as neutrophil reverse migration. Here we apply microscale organotypic models to analyze onset and resolution of inflammation by 1) analyzing migration and signaling through 3D co-culture of primary neutrophils or induced pluripotent stem cells (iPSCs) derived neutrophils, 2) spatiotemporally separating and retrieving neutrophils during forward and reverse migration and 3) modeling neutrophil migration and reverse migration in a physiologically relevant 3D microenvironment, and. It is our goal to develop a physiologically relevant in vitro model to replicate key steps in neutrophil recruitment to and clearance from a site of inflammation. A key strength in this application is the use of organotypic microscale models that allow for the replication of the essential geometries and cellular interactions that induce and resolve neutrophil inflammation. A broad goal for the proposed research is to provide a collaborative, multi-disciplinary (engineering, biologists, clinicians) approach to a fundamental problem relevant to human health ? namely, inflammation and its resolution at sites of tissue injury. We strive to develop practical tools and methods that will transform our ability to identify signaling pathways that modify neutrophil forward and reverse migration and have therapeutic potential. A particular focus is the paracrine signals generated by macrophages, endothelial cells, and neutrophils in co- and tri-culture and the characterization of induced pluripotent stem cell derived neutrophils as a model for primary human neutrophils (Aim 1). Furthermore, the use of layered open microfluidics technology will allow us to separate neutrophils at different stages in bidirectional migration to identify spatiotemporal regulation of neutrophil behavior (Aim 2). Finally, we will investigate the trafficking of human neutrophils and their interactions with macrophages and endothelial cells in response to different inflammatory stimuli in a physiologically relevant organotypic in vitro model to examine neutrophil reverse migration and how it is altered in disease (Aim 3). We expect our findings to address a gap in understanding how cells communicate during the onset and resolution of inflammation, and the further development of iPS derived neutrophils that may provide novel avenues for therapeutic interventions for human disease.